Preparation of 2-(4-hydroxyphenoxy)propionic acid by fermentation

ABSTRACT

A process for the oxidation of 2-phenoxypropionic acid to 2-(4-hydrophenoxy)propionic acid using microorganisms is described.

The present invention relates to a process for the preparation of 2-(4-hydroxyphenoxy)propionic acid.

2-(4-Hydroxyphenoxy)propionic acid is a valuable intermediate, in particular for the preparation of herbicides.

Chemical processes for the preparation of racemic and of pure enantiomeric forms of 2-(4-hydroxyphenoxy)propionic acid have been disclosed. These processes start from hydroquinone and have the disadvantage that they give by-products whose removal, which is necessary, is laborious. This makes economic preparation impossible.

It has been disclosed that microorganisms are able to hydroxylate aromatic compounds. This usually produces dihydroxylated compounds or mixtures of various regioisomers (R. J. W. Byrde, D. Woodcook, Biochem. J. 65 (1957) 682).

The regioselective oxidation of 2-phenoxypropionic acid to 2-(4-hydroxyphenoxy)propionic acid by microorganisms has not hitherto been disclosed.

We have now found, surprisingly, that various microorganisms oxidize the valuable 2-phenoxypropionic acid to 2-(4-hydroxyphenoxy)propionic acid with high regioselectivity.

The present invention relates to a process for the preparation of 2-(4-hydroxyphenoxy)propionic acid by fermentation, which comprises oxidizing 2-phenoxypropionic acid or salts thereof under aerobic conditions in the presence of a microorganism.

A large number of microorganisms are suitable for the process, in particular fungi and bacteria. Utilizable fungi can be isolated, for example, from soil samples, in particular humus-containing soil samples. Particularly suitable for the reaction are Aspergillus niger strains. Also suitable are many fungi from the genera Beauveria, Paecilomyces, Sclerotium and Coprinus, which can be obtained, for example, from collections of strains and whose suitability for the hydroxylation can be determined in a straightforward preliminary test. Suitable bacteria can also be isolated from soil samples, inter alia. Particularly suitable bacteria are Streptomyces strains.

To carry out the process according to the invention, suitable strains are transferred to a nutrient medium containing 2-phenoxypropionic acid and incubated aerobically therein under conditions favorable for growth and production by the particular microorganism. The fermentation is carried out continuously or batchwise for 1 to 10 days.

The cells of the microorganism, which can also be used in the form of dormant, non-growing cells, are allowed to act directly on the substrate. All conventional incubation processes can be employed, but particularly preferred fermenters are in the form of deep, aerated and agitated tanks. Very good results are obtained by incubation of a liquid nutrient medium.

Suitable nutrient media contain sources of carbon and of nitrogen, and inorganic salts and, where appropriate, small amounts of trace elements and vitamins. Sources of nitrogen which can be used are inorganic or organic nitrogen compounds or materials which contain these compounds. Examples are ammonium salts, nitrates, corn steep liquor, brewer's yeast autolysate, soybean meal, wheat gluten, yeast extract, yeast, urea and potato protein. Examples of sources of carbon which can be used are sugars such as glucose, polyols such as glycerol, or fats such as soybean oil.

Examples of inorganic salts are the salts of calcium, magnesium, manganese,, potassium, zinc, copper, iron and other metals. A particularly suitable anion in the salts is the phosphate ion. Growth factors may be added to the nutrient medium, such as biotin, riboflavin or other vitamins.

The ratios of the said nutrients in the mixture depends on the type of fermentation and will be determined in the individual case.

Generally suitable for carrying out the process according to the invention are phenoxypropionic acid concentrations of about 1 to 100 g/l, preferably about 5 to 50 g/l.

The culture conditions are chosen so that the best possible yields are achieved. Cultivation is preferably carried out at from 20° to 40° C., particularly advantageously from 25° to 30° C. The pH is preferably maintained in the range 3 to 9. The pH is particularly advantageously from 4 to 7. An incubation time of from 15 to 100 hours generally suffices. The maximum amount of the desired product accumulates in the medium during this time. The acid is preferably added in the form of a salt, e.g. the sodium salt. The acid can be added to the nutrient medium all at once at the start, after growth has taken place or in several portions or continuously during the cultivation.

The use of 2-phenoxypropionic acid is preferred, but it is also possible to use salts thereof. Preferred salts are alkali metal and alkaline earth metal salts, for example the Na, K and Li salts.

The novel process is suitable for the oxidation both of racemic 2-(4-hydroxyphenoxy)propionic acid and of antipodes thereof. The center of asymmetry remains untouched in the process according to the invention. If, for example, R-2-phenoxypropionic acid is used, the configuration is retained and the product is R-2-(4-hydroxyphenoxy)propionic acid. No other hydroxylation products are found in the reaction. Thus the novel process represents a straightforward and more economic method for the selective preparation of 2-(4-hydroxyphenoxy)propionic acid.

The examples which follow illustrate the invention:

EXAMPLE 1

Two liquid nutrient media were used to examine various strains of bacteria:

    ______________________________________                                         Medium A                                                                       ______________________________________                                         20      g/l       glucose                                                      5       g/l       yeast extract                                                5       g/l       ammonium sulfate                                             0.5     g/l       magnesium sulfate 7-hydrate                                  0.05    g/l       manganese sulfate 1-hydrate                                  1.5     g/l       potassium dihydrogen phosphate                               3.6     g/l       dipotassium hydrogen phosphate                               1       g/l       (R)-2-phenoxypropionic acid                                  2       mg/l      iron(II) sulfate 1-hydrate                                   100     μg/l   zinc(II) sulfate 4-hydrate                                   300     μg/l   boric acid                                                   200     μg/l   cobalt(II) chloride 6-hydrate                                10      μg/l   copper(II) chloride 2-hydrate                                20      μg/l   nickel(II) chloride 6-hydrate                                30      μg/l   sodium molybdate 2-hydrate                                   ______________________________________                                    

The pH was adjusted to 6.8 with 5N sodium hydroxide solution. Glucose and phosphates were each autoclaved separately at 121° C. for 10 minutes. The (R)-2-phenoxypropionic acid was dissolved in water, with a little 5N sodium hydroxide solution, and sterilized by filtration. The remaining medium was autoclaved at 121° C. for 10 minutes.

    ______________________________________                                         Medium B                                                                       ______________________________________                                         20      g/l      glucose                                                       40      g/l      corn steep liquor                                             1.5     g/l      potassium dihydrogen phosphate                                3.6     g/l      dipotassium hydrogen phosphate                                1       g/l      (R)-2-phenoxypropionic acid                                   ______________________________________                                    

The pH was adjusted to 6.8 with 5N sodium hydroxide solution. Glucose and phosphates were each autoclaved separately at 121° C. for 10 minutes. The (R)-2-phenoxypropionic acid was dissolved in water, with a little 5N sodium hydroxide solution, and sterilized by filtration. The remaining medium was autoclaved at 121° C. for 45 minutes.

20 ml portions of the sterile medium were introduced into sterile 100 ml Erlenmeyer flasks, which were provided with a sterile cotton plug. One loop of the strain of bacteria to be tested was used to inoculate each flask. The flasks were then incubated at 28° C., shaking at 250 rpm, for 3 to 7 days. Then 1000 μl of fermentation broth were removed, 100 μl of 1N HCl and 800 μl of ethyl acetate were added, and the mixture was vigorously mixed for 15 seconds. 700 μl of the organic phase were carefully removed and evaporated in a test tube under a gentle stream of nitrogen at 50° C. The residue was dissolved in 70 μl of ethyl acetate and transferred quantitatively into a test tube for the gas chromatography. To this were added 30 μl of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA). The samples were then examined by gas chromatography. The external standard used was an authentic sample of (R,S)-2-(4 -hydroxyphenoxy)propionic acid which had been chemically synthesized.

The table which follows summarizes the results:

    ______________________________________                                                     Collection/ Conversion                                             Genus/species                                                                              Number      (%)        Medium                                      ______________________________________                                         Streptomyces                                                                               DSM 40725   11         A                                           antibioticus                                                                   Streptomyces                                                                               ATCC 11891  10         A                                           antibioticus                                                                   Streptomyces                                                                               ATCC 13850  18         A                                           flocculus                                                                      Streptomyces                                                                               ATCC 19040  97         B                                           hygroscopicus                                                                  Streptomyces                                                                               ATCC 21705  98         A                                           hygroscopicus                                                                  Streptomyces                                                                               ATCC 15715   4         A                                           kasugaensis                                                                    Streptomyces                                                                               ATCC 15714  23         B                                           kasugaensis                                                                    Streptomyces                                                                               ATCC 13279  13         A                                           mediocidicus                                                                   Streptomyces                                                                               ATCC 19793  23         A                                           niveus                                                                         Streptomyces                                                                               ATCC 31055   2         A                                           panayensis                                                                     Streptomyces                                                                               ATCC 21895   4         A                                           roseochromogenes                                                               Streptomyces                                                                               ATCC 11989  24         A                                           viridifaciens                                                                  ______________________________________                                    

EXAMPLE 2

About 400 different novel Streptomycetes strains were isolated from various soil samples by the method of DREWS (Mikrobiologisches Praktikum, 3rd edition, Springer Verlag, pages 47-48, 1976). These strains were tested by the method of Example 1. The table which follows summarizes the conversion by the strains identified as positive.

    ______________________________________                                         Strain number Conversion (%)                                                                             Medium                                               ______________________________________                                         1034           8          A                                                    1045          28          A                                                    1053          22          A                                                    1057           7          A                                                    1071          11          A                                                    2476          13          A                                                    3279           4          A                                                    3292          33          B                                                    3388          26          B                                                    3520          42          B                                                    3523          80          A                                                    3549          80          B                                                    3558          24          A                                                    3559          32          A                                                    3560          98          A                                                    3561          90          A                                                    3565           4          A                                                    3567          72          B                                                    3574          41          A                                                    3575          24          B                                                    3782          44          A                                                    3925          24          B                                                    4041          14          A                                                    5431          19          A                                                    5432          99          A                                                    5607          24          A                                                    5608          26          A                                                    5613          33          A                                                    5619          51          A                                                    5632          18          A                                                    5635          15          A                                                    5636           5          A                                                    5637          39          A                                                    5638          38          A                                                    5648          25          A                                                    5654          59          A                                                    ______________________________________                                    

EXAMPLE 3

A liquid nutrient medium was prepared with the following ingredients:

    ______________________________________                                         20     g/l     glucose                                                         5      g/l     yeast extract                                                   5      g/l     ammonium sulfate                                                0.5    g/l     magnesium sulfate 7-hydrate                                     0.05   g/l     manganese sulfate 1-hydrate                                     1.5    g/l     potassium dihydrogen phosphate                                  3.6    g/l     dipotassium hydrogen phosphate                                  3      g/l     Carbopol ® 946                                                             (Carboxyvinyl polymer with an extremely                                        high molecular weight)                                          3      g/l     (R)-2-phenoxypropionic acid                                     2      mg/l    iron(II) sulfate 1-hydrate                                      100    μg/l zinc(II) sulfate 4-hydrate                                      300    μg/l boric acid                                                      200    μg/l cobalt(II) chloride 6-hydrate                                   10     μg/l copper(II) chloride 2-hydrate                                   20     μg/l nickel(II) chloride 6-hydrate                                   30     μg/l sodium molybdate 2-hydrate                                      ______________________________________                                    

The pH was adjusted to 6.8 with 5N sodium hydroxide solution. Glucose and phosphates were each autoclaved separately at 121° C. for 10 minutes. The (R)-2-phenoxypropionic acid was dissolved in water with a little 5N sodium hydroxide solution and sterilized by filtration. The remaining medium was autoclaved at 121° C. for 10 minutes. 20 ml portions of the sterile medium were introduced into sterile 100 ml Erlenmeyer flasks, which were provided with a sterile cotton plug. One loop of spores of the Aspergillus niger ATCC 11394 fungus strain was used to inoculate one flask. The flask was then incubated at 28° C., shaking at 250 rpm, for three days. Then 1000 μl of fermentation broth were removed, 100 μl of 1N HCl and 800 μl of ethyl acetate were added, and the mixture was vigorously mixed for 15 seconds. 700 μl of the organic phase were carefully removed and evaporated in a test tube under a gentle stream of nitrogen at 50° C. The residue was dissolved in 70 μl of ethyl acetate and transferred quantitatively into a test tube for the gas chromatography. To this were added 30 μl of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA). The sample was then examined by gas chromatography. An authentic sample of 2-)(4-hydroxyphenoxy)-propionic acid was sued as reference.

EXAMPLE 4

A liquid nutrient medium was prepared with the following ingredients:

    ______________________________________                                         40      g/l       glucose                                                      10      g/l       yeast extract                                                0.5     g/l       magnesium sulfate 7-hydrate                                  0.05    g/l       manganese sulfate 1-hydrate                                  1.5     g/l       potassium dihydrogen phosphate                               3.6     g/l       dipotassium hydrogen phosphate                               20      g/l       (R)-2-phenoxypropionic acid                                  2       mg/l      iron(II) sulfate 1-hydrate                                   100     μg/l   zinc(II) sulfate 4-hydrate                                   300     μg/l   boric acid                                                   200     μg/l   cobalt(II) chloride 6-hydrate                                10      μg/l   copper(II) chloride 2-hydrate                                20      μg/l   nickel(II) chloride 6-hydrate                                30      μg/l   sodium molybdate 2-hydrate                                   ______________________________________                                    

The pH was adjusted to 6.8 with 5N sodium hydroxide solution. Glucose and phosphates were each autoclaved separately at 121° C. for 10 minutes. The (R)-2-phenoxypropionic acid was dissolved in water with a little 5N sodium hydroxide solution and sterilized by filtration. The remaining medium was autoclaved at 121° C. for 10 minutes. 50 ml of the sterile medium were introduced into a sterile 100 ml Erlenmeyer flask, which was provided with a sterile cotton plug.

After inoculation wit 2.5 ml of a 72 h-old preculture of Beauveria bassiana ATCC 7159 (cultured in the same medium but in the presence of only 5g/l (η)-2-phenoxypropionic acid), the mixture was incubated at 28° C., shaking at 200 rpm, for 4-5 days. The subsequent analysis by gas chromatography revealed that the acid had been 98% hydroxylated.

98% of the (R)-2-phenoxypropionic acid had been oxidized to (R)-2-(4-hydroxyphenoxy)propionic acid. The result was the same when the S enantiomer or the racemate of 2-phenoxypropionic acid was employed.

EXAMPLE 5

A novel strain of Aspergillus niger was isolated from a humus-containing soil sample by the method of DREWS (Mikrobiologisches Praktikum, 3rd edition, Springer Verlag, pages 51-53 (1976)). This strain (No. 1777) was tested by the method of Example 3. 99.8% of the acid had been oxidized after 3 days.

EXAMPLE 6

Further strains of the species Aspergillus niger were tested as described in Example 3 and analyzed after fermentation for 3 and 7 days. The results are summarized in the table which follows:

    ______________________________________                                                      Collection/  Conversion                                           Genus/species                                                                               Number       (%)       Days                                       ______________________________________                                         Aspergillus niger                                                                           ATCC 9142    34        3                                                       ATCC 11394   98        3                                          Aspergillus niger                                                                           ATCC 26693   100       3                                          Aspergillus niger                                                                           ATCC 1015    83        3                                          Aspergillus niger                                                                           ATCC 10577   70        3                                          Aspergillus niger                                                                           ATCC 13794   99        3                                          Aspergillus niger                                                                           ATCC 11414   79        3                                          Aspergillus niger                                                                           ATCC 26550   79        3                                          Aspergillus niger                                                                           ATCC 9029    71        3                                          Aspergillus niger                                                                           ATCC 9642    41        3                                          Aspergillus niger                                                                           ATCC 32656   27        7                                          Aspergillus niger                                                                           ATCC 6275    11        3                                          Aspergillus niger                                                                           ATCC 16404   97        3                                          Aspergillus niger                                                                           ATCC 10575   53        7                                          Aspergillus niger                                                                           ATCC 10581   85        3                                          Aspergillus niger                                                                           ATCC 10549   27        7                                          Aspergillus niger                                                                           ATCC 1027    51        7                                          Aspergillus niger                                                                           ATCC 1040    97        7                                          Aspergillus niger                                                                           ATCC 10553   14        7                                          Aspergillus niger                                                                           ATCC 10864   65        7                                          Aspergillus niger                                                                           ATCC 1004    79        7                                          Aspergillus niger                                                                           ATCC 16880   99        7                                          Aspergillus niger                                                                           ATCC 16888   54        7                                          Aspergillus niger                                                                           ATCC 6273    89        7                                          Aspergillus niger                                                                           ATCC 7797    79        7                                          Aspergillus niger                                                                           ATCC 7983    90        7                                          Aspergillus niger                                                                           ATCC 10574   100       7                                          Aspergillus niger                                                                           ATCC 10578   100       7                                          ______________________________________                                    

EXAMPLE 7

Further fungi were tested as in Example 3 and analyzed after fermentation for 3 and 7 days.

    ______________________________________                                                       Collection/ Conversion                                           Genus/species Number      (%)       Days                                       ______________________________________                                         Aspergillus   ATCC 8740    7        7                                          carbonarius                                                                    Aspergillus   ATCC 6276    5        7                                          carbonarius                                                                    Aspergillus   ATCC 10254  62        7                                          foetidus                                                                       Aspergillus   ATCC 26850   7        3                                          parasiticus                                                                    Aspergillus   DSM 63357    5        3                                          sclerotiorum                                                                   Aspergillus   CMI 112328  17        3                                          sclerotiorum                                                                   Aspergillus   CMI 116935  15        3                                          sclerotiorum                                                                   Beauveria bassiana                                                                           ATCC 7159   99        3                                          Paecilomyces  ATCC 26853  63        7                                          farinosus                                                                      Sclerotium rolfsii                                                                           ATCC 15206  85        7                                          Sclerotium rolfsii                                                                           ATCC 15204  19        7                                          Sclerotium rolfsii                                                                           ATCC 15203  89        7                                          Sclerotium rolfsii                                                                           ATCC 15201  81        7                                          Sclerotium rolfsii                                                                           ATCC 15195  85        7                                          Sclerotium rolfsii                                                                           ATCC 26326   4        7                                          Sclerotium    ATCC 15196   1        7                                          delphinii                                                                      Coprinus cinereus                                                                            ATCC 20120   2        7                                          ______________________________________                                     

We claim:
 1. A process for the preparation of 2-(4-hydroxyphenoxy)propionic acid by fermentation, wherein hydroxylation takes place substantially only in the p-position, which process comprises oxidizing 2-phenoxypropionic acid of salts thereof under aerobic conditions in the presence of a 2-(4-hydroxyphenoxy) propionic acid activity-producing, soil-inhabiting microorganism selected from the group consisting of a Streptomycetes bacteria and a fungus.
 2. The process of claim 1 wherein the microorganism is an Aspergillus niger fungus.
 3. The process of claim 1 wherein the microorganism is a Streptomycetes bacterium.
 4. A process as defined in claim 1 wherein the enantiomers of 2-phenoxypropionic acid are employed.
 5. The process of claim 1, wherein the microorganism is a fungus selected from the group consisting of Beauveria bassiana, Paecilomyces farinosus, Sclerotium folfsii, Sclerotium delphinii, and Coprinus cinereus. 